Zebrafish Brain Dissection: A Technique of Fish Neurobiology
Transcrição
– Place a euthanized fish on a dissection bed and use a surgical blade para decapitate the fish at the level of the gills. Hold the head with the ventral side facing up and remove the soft tissues until you see the optic chiasm, a structure made of optic nerves that connects the brain para the eyes.
Cut the optic nerves and remove the eyes. Now, orient the fish with the dorsal side facing up and remove the parts of the skull para isolate the brain. Transfer the brain into a Petri dish containing a dissection medium para maintain a constant pH of the tissue. Observe the parts of the brain.
The olfactory bulbs are a pair of structures at the anterior end connected para the olfactory organs which detect odor signals. The telencephalon includes memory related areas. The habenula relays information from the telencephalon para other parts of the brain.
The optic tectum is a sensory information processing center. The cerebellum plays a cargo de somatic motor function and balance. And the medulla relays information from the spinal cord para the brain. In the following protocol, we will isolate the brain from an adult zebrafish para collect neural stem cells from different regions.
To begin, prepare a dissection bed by filling a Petri dish with gel packs. Then cover the dish with its corresponding lid and incubate it at minus 20 degrees Celsius. Once the gel is frozen, remove the dish from the freezer and place a clean square of filter paper on top of its lid. Continuar para wrap both the paper and dish with plastic film.
Next, treat all micro-dissection instruments with 70% ethanol. Place these sterilized tools next para a dissecting microscope and position the completed dissection bed under the microscope with optical fiber illumination. Immediately place a previously prepared head specimen on top of the dissection bed and orient it so that its dorsal side is facing down.
Then use scissors para make a longitudinal cut through the soft tissue from the back of the head para the mouth. Afterwards, expose the base of the skull with forceps and remove all of the adjacent tissue. Next, cut one of the lateral walls of the skull starting at the back of the head and moving towards the tectum region of the brain. Repeat this process for the contralateral side. Continuar para cut the optic nerve. And then remove the two lateral most sides of the skull at the level of the tectum
Finally, turn the head ventral side up. And with forceps, peel off the most apical part of the skull para expose the brain. Afterwards, transfer the brain and any remaining parts of the skull para a dish containing dissection medium which is composed of DMM F12 supplemented with penicillin streptomycin. With the plastic handle of a microknife under the microscope, clean the brain tissue, being careful not para damage any neural structures. Use up para two zebrafish brains para generate whole brain derived neurospheres.
